Coccidiosis poultry vaccine

ABSTRACT

This invention relates to novel Eimeria proteins with immunogenic properties as well as to DNA sequences encoding these proteins. These proteins can be administered to poultry thereby protecting the birds against coccidiosis. In addition the DNA encoding these proteins can be used for the preparation of a vector vaccine against coccidiosis.

This is a continuation, of application Ser. No. 08/338,057 filed Nov.10, 1994.

FIELD OF THE INVENTION

The present invention relates to a protein derived from an Eimeriaspecies, in particular Eimeria maxima, which is capable of stimulatingimmune lymphocytes. It also relates to a nucleic acid sequence encodingall or an antigenically significant part of this protein, a recombinantvector comprising such a nucleic acid sequence, a host cell or organismtransformed with such a recombinant vector and a vaccine for theprotection of poultry against coccidiosis.

BACKGROUND OF THE INVENTION

Coccidiosis is a disease caused by infection with one or more of themany species of coccidia, intracellular protozoal parasites of thesubphylum Apicomplexa and the genus Eimeria. Poultry is defined hereinas domesticated birds that serve as a source of eggs or meat and thatinclude such commercially important kinds as chickens, turkeys, ducks,geese, guinea fowl, pheasants, pigeons and peafowl.

Coccidiosis in chickens is known to be caused by several differentspecies of Eimeria, namely Eimeria acervulina, E. maxima, E. tenella, E.necatrix, E. brunetti, E. mitis, E. praecox, E. mivati and E. hagani.Some people, however, doubt the true existence of the last two species.Low level infection with any of these Eimeria species results in aprotective immunity to reinfection.

The species do differ in their pathogenic effect on chickens, the typeof chicken also playing a role; thus, a broiler chicken will besubjected to a great deal of damage by a parasite such as E. acervulinaor E. maxima because these parasitise large portions of the smallintestine, where food digestion plays a major role.

E. maxima is the most immunogenic of the species listed above, producinggood natural protection following infection. There are, however, strainvariations with little or no cross protection between strains.

During the life cycle, the Eimeria parasite passes through a number ofstages. The life cycle begins when the chicken ingests the infectiousstage, known as the sporulating oocyst, during ground feeding or byinhalation of dust. In the case of E. maxima, the oocyst is unusuallylarge. The wall of the sporulated oocyst is ruptured by a combination ofmechanical grinding action and chemical action in the gizzard andintestinal tract, resulting in the release of four sporocysts. Thesporocysts pass into the duodenum where they are exposed to bile anddigestive enzymes resulting in the release of an average of tensporozoites per sporocyst.

The sporozoites are mobile and search for suitable host epithelium cellsin order to penetrate and reproduce in them. Following infection of anepithelium cell, the parasite enters the schizont phase of its lifecycle, producing from 8 to 16 to >200 merozoites per schizont. Oncereleased from the schizont, the merozoites are free to infect furtherepithelium cells. After from two to five of these asexual reproductioncycles, the intracellular merozoites grow into sexual forms known as thefemale or macrogametocyte and the male or microgametocyte. Followingfertilization of the macrogametocyte by the microgametes released fromthe microgametocyte, a zygote is formed which creates a cyst wall aboutitself. The newly formed oocyst is passed out of the infected chickenwith the droppings.

With the correct environmental conditions of temperature and humidityand sufficient oxygen in the air, the oocyst will sporulate into theinfectious stage, ready to infect a new host and thereby spreading thedisease. Thus no intermediate host is required for transfer of theparasite from bird to bird.

The result of the Eimeria parasite infecting the digestive tract of achicken may be a reduction in weight gain, decreased feed conversion,cessation of egg production and, in many cases, death. The increase inintensive production of poultry has been accompanied by severe lossesdue to this parasite; indeed, coccidiosis has become the mosteconomically important parasitic disease. In the Netherlands, the lossesthat poultry farmers suffer every year run into millions of guilders; in1986 the loss was about 13 million guilders. In the same year, a loss of300 million dollars was suffered in the United States.

In the past, several methods have been used in attempts to controlcoccidiosis. Prior to the advent of chemotherapeutic agents, improvedsanitation using disinfectants, together with the mechanical removal oflitter, was the main method employed; sufficient oocysts, however,usually remained to transmit the disease.

The introduction of coccidiostatic agents in the feed or drinking water,in addition to good management, resulted in some success at diseasecontrol. Such agents have been found to suffer from a drop ineffectiveness over the years, due partly to the development of drugresistant strains of coccidia. Furthermore, several chemotherapeuticagents have been found to leave residues in the meat, making itunsuitable for consumption.

Attempts have been made to control the disease immunologically byadministering to chickens a live vaccine comprising oocysts from allseven species of Eimeria, the oocysts administered being from precociouslines. Such precocious lines are obtained by inoculating chickens with awild population of an Eimeria species and collecting the very firstparasites that are excreted as a result of the infection. The collectedparasites are put back into chickens and the cycle is repeated severaltimes. Eventually a precocious line of parasite is produced which hasfewer cycles of asexual reproduction in the gut. Thus such lines retaintheir immunogenicity, whilst producing fewer parasites in the gut withless consequential damage being caused to the host chicken. Thedisadvantage of this type of vaccine is that it is expensive to producebecause of the necessity of producing it in live chickens and its lowerreproductive potential.

The advent of genetic engineering has provided new methods for producingeffective vaccines. Using these methods, the DNA coding for theantigenic proteins of some pathogenic microorganisms has been clonedinto such host microorganisms as Escherichia coli, with the result thatthe protein has been expressed at sufficiently high levels such that itcan be incorporated into a vaccine. The advantage of proteins producedin this way is that they are noninfectious and are relatively cheap toproduce. In this way, vaccines have been prepared against a number ofviruses such as hepatitis, herpes simplex and foot and mouth disease.

Attempts have been made to genetically engineer a coccidiosis vaccine.European patent application No. 337 589 describes the isolation of aGroup B Eimeria tenella protein and its insertion into a novelexpression vector which, in turn, has been used to transform appropriatehosts. Patent Cooperation Treaty Application WO 92/04461 describes theconstruction of a microorganism that produces an antigenic protein usingeither the "mRNA route" or the "nuclear DNA route". In this way, certainantigens from E. tenella and E. maxima were prepared and sequenced.Taking this type of route to prepare antigens for incorporation into avaccine relies only upon selecting antigens which could induceantibodies in an heterologous species. This approach does notnecessarily end up with selecting the most protective antigen.

From H. S. Lillehoj (Vet. Immunol. Immunopath., 13, 321-330, 1986) itcan be conceived that development of protective immunity in chickensinfected with coccidia may be due to the development of aspecies-specific T cell response.

SUMMARY OF THE INVENTION

It has now been found that by fractionating Eimeria parasites andselecting proteins that stimulate immune T-lymphocytes, then preparingvectors containing the nucleic acid coding for such proteins andsubsequently preparing a vaccine containing such proteins, a moreeffectively protective coccidiosis vaccine may be produced.

According to one aspect of the present invention, there is provided apurified Eimeria maxima T-lymphocyte stimulatory protein or animmunogenically active part thereof. Such a protein is essentially freefrom the whole parasite or other proteins with which they are ordinarilyassociated.

According to a second aspect of the invention, there is provided anucleic acid sequence encoding all or a substantial part, in particularthe immunologically active part, of a purified Eimeria maximaT-lymphocyte stimulatory protein. Such a nucleic acid sequence may beoperatively linked to expression control sequences resulting in arecombinant nucleic acid molecule which, when inserted into a suitablevector, results in a recombinant vector capable of expressing thenucleic acid sequence.

Such a recombinant vector, or nucleic acid sequence as defined above,may be used to transform a suitable host cell or organism. Such atransformed host cell or organism may, in turn, be used to produce thestimulatory protein for incorporation into a vaccine for the protectionof poultry against coccidiosis. Alternatively, the transformed host cellor organism may itself be incorporated into a vaccine.

In general, the term "protein" refers to a molecular chain of aminoacids with biological activity. A protein is not of a specific lengthand can, if required, be modified in vivo or in vitro, by, for example,glycosylation, amidation, carboxylation or phosphorylation; thus, interalia, peptides, oligopeptides and polypeptides are included within thedefinition.

More particularly, this invention provides T-lymphocyte stimulatoryproteins, or immunogenically active parts thereof, which have the aminoacid sequence shown in SEQ ID NO. 2 and 4 and their biologicallyfunctional equivalents or variants.

The biologically functional equivalents or variants of the proteinsspecifically disclosed herein are proteins derived from the abovenotedamino acid sequences, for example by deletions, insertions and/orsubstitutions of one or more amino acids, but retain one or moreimmunogenic determinants of the Eimeria antigens, i.e. said variantshave one or more epitopes capable of eliciting an immune response in ahost animal.

It will be understood that, for the particular proteins embraced herein,natural variations can exist between individual Eimeria parasites orstrains. These variations may be demonstrated by (an) amino aciddifference(s) in the overall sequence or by deletions, substitutions,insertions, inversions or additions of (an) amino acid(s) in saidsequence. Amino acid substitutions which do not essentially alterbiological and immunological activities, have been described, e.g. byNeurath et al in "The Proteins" Academic Press New York (1979). Aminoacid replacements between related amino acids or replacements which haveoccurred frequently in evolution are, inter alia, Ser/Ala, Ser/Gly,Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequenceand structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5,suppl. 3). Other amino acid substitutions include Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Thr/Phe, Ala/Pro, Lys/Arg, Leu/Ile,Leu/Val and Ala/Glu. Based on this information, Lipman and Pearsondeveloped a method for rapid and sensitive protein comparison (Science,227, 1435-1441, 1985) and determining the functional similarity betweenhomologous proteins. Such amino acid substitutions of the exemplaryembodiments of this invention are within the scope of the invention aslong as the resulting proteins retain their immunoreactivity.

The invention further provides isolated and purified nucleic acidsequences encoding the above mentioned proteins of Eimeria. Such nucleicacid sequences are shown in SEQ. ID. NOS. 1,3 and 5. It is well known inthe art that the degeneracy of the genetic code permits substitution ofbases in the codon resulting in another codon but still coding for thesame amino acid, e.g. the codon for the amino acid glutamic acid is bothGAT and GAA. Consequently, it is clear that, for the expression of aprotein with the amino acid sequence shown in SEQ. ID. NOS. 2 and 4, thenucleic acid sequence may have a codon composition different from thenucleic acid sequence shown in SEQ. ID. NOS. 1 and 3, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Western blot of E. maxima sporozoites screened withanti-70kD sera.

FIG. 2 shows the restriction maps of p70 cDNA inserts (5'-3').

FIG. 3 shows a Western blot of purified pEm 70/1 protein probed withchicken anti-recombinant pEm 70/1 serum.

FIG. 4 is an SDS-PAGE of the purification of recombinant pEm 70/1.

FIG. 5 is the genetic map of pMLB1113(His)6-3.

DETAILED DESCRIPTION OF THE INVENTION

Eimeria maxima parasites were produced by passage through chickens asdescribed by Long et al (Folio Vet. Lat., 1976, 6, 201-207). Oocystswere isolated from the faeces of the infected chickens, sporulated andthen purified by floatation in saturated sodium chloride. The sporulatedoocysts were then used to infect pathogen-free 4 week old chickens. Afurther dose of sporulated oocysts was administered to the birds inorder to boost their immune response.

A preparation of sporulated oocysts purified by floatation as describedabove was subjected to vibration in a disintegrator in order to releasesporozoites. The sporozoites were treated with proteolytic enzymes inorder to release sporozoites which were then washed and purified byion-exchange chromatography according to the method of Schmatz et al (J.Protozool., 1984, 31, 181-183). The sporozoites were suspended inbuffer, boiled in a water bath and spun prior to loading on apolyacrylamide gel for SDS-PAGE(sodium dodecyl sulphate polyacrylamidegel electrophoresis). Molecular mass markers were run on the same gel inorder to extrapolate the molecular mass of the Eimeria antigens. The gelwas electrophoresed onto nitrocellulose paper by the method of Towbinand Gordon, (J. Immunol. Methods., 1984, 72, 313-340). Thenitrocellulose paper was then washed and visualised by Aurodye stainingaccording to the manufacturer's instructions.

Protein bands were excised from the nitrocellulose paper, cut into smallpieces and transferred to glass vials. The nitrocellulose pieces werethen solubilised in dimethyl sulphoxide (DMSO) and left for a period oftime to ensure solubilization, after which the nitrocellulose particleswere precipitated by dropwise addition of carbonate/bicarbonate bufferwith vigorous vortexing. Samples were then centrifuged, the pellets ofnitrocellulose particles were washed several times after which they wereresuspended and divided into small aliquots.

In order to determine whether any of the protein bands from theelectrophoresis gel stimulated the lymphocytes from infected birds,blood was withdrawn by venopuncture from the chickens infected asdescribed above, for use in a lymphocyte proliferation assay. The bloodwas centrifuged at 600 g, after 10 minutes the suspension of cells abovethe sedimented erythrocytes was removed and centrifuged at 400 g for afurther 10 minutes. The cells deposited after the second centrifugationwere washed several times and finally resuspended. The resuspended cellswere cultured in round bottomed plates together with the dilutedresuspended nitrocellulose particles. Control wells were set upcontaining nitrocellulose particles devoid of protein.

The lymphocyte cultures were incubated for 96 hours, during the last 16hours of which the cultures were pulsed with 3H-thymidine, followingwhich the cells were harvested onto glass microfibre filters. Afterdrying, the filters were placed in scintillation vials to which wasadded liquid scintillation cocktail (Scintillator 299 registered trademark! Packard, Caversham, U.K.) and the radioactivity was measured in ascintillation spectrophotometer. The results were expressed as astimulation index (SI) obtained using the following formula:

    SI=cpm1/cpm2

where:

cpm1=average counts per minute of triplicate cultures incubated with NCparticles bearing protein.

cpm2=average counts per minute of triplicate cultures incubated with NCparticles devoid of protein.

By this method mostly T-lymphocytes are proliferating.

The results showed that although the stimulation index varied fordifferent birds and different gels, a protein band, with a relativemolecular mass (Mr) of approximately 70.000 D, gave consistantstimulation of lymphocytes from immunised but not control birds.

Following this discovery, a fresh preparation of E. maxima sporozoiteswas separated by SDS-PAGE and transferred to nitrocellulose as describedabove. A protein band with a Mr of 70.000 D (p70) was excised,solubilised as described, washed in phosphate buffered saline (PBS) andthen resuspended in PBS. This suspension was inoculated subcutaneouslyinto rabbits, the injections being repeated every 2 weeks. Two weeksafter each injection the rabbits were bled by venapuncture of a lateralear vein. Rabbit anti-p70 serum was obtained after 5 boosts asdetermined by Western blotting.

Total ribonucleic acid (RNA) was extracted and purified from E. maximasporozoites by centrifugation through a gradient of cesiumtrifluoroacetate. In order to separate the messenger RNA (mRNA) fromnon-mRNA, columns of oligo dT CELLULOSE (poly A! Quik, Stratagene) wereused according to the manufacturer's instructions. The poly(A)+ RNA ormRNA was then eluted from the column overnight using sodium acetate inabsolute ethanol.

Copy deoxyribonucleic acid (cDNA) was synthesised from the mRNA using aZAP-cDNA (registered trade mark) synthesis kit (Stratagene). The firststrand of cDNA was synthesised using an oligo dT template (containing anXhoI restriction site) and Moloney-Murine Leukaemia Virus reversetranscriptase. The cytosine residues in the first strand of cDNA weremethylated in order to protect the cDNA from digestion by restrictionenzymes to be used later in the cloning protocol. The second strand ofcDNA was synthesised using RNAse H and DNA polymerase I followed byend-repairing using T4 DNA polymerase. EcoRI adapters were ligated tothe blunt ended cDNA by T4 DNA ligase. Digestion with XhoI produced cDNAwith an XhoI compatible 3' end and an EcoRI compatible 5' end.

The cDNA was ligated to EcoRI/XhoI digested and dephosphorylated Uni-ZAPXR vector using T4 DNA ligase. The resulting primary libraries (Emx 8and Emx 9) were plated and amplified on E. coli SURE cell. It was foundthat the Emx8 library gave 65% recombinants, whereas the Emx9 librarygave 55% recombinants.

The two libraries, Emx8 and Emx9, were screened using rabbit anti-p70serum, prepared as described above. Positive plaques were picked out,and rescreened until the positives were plaque pure.

The cDNA from clones in the two libraries, Emx8 and Emx9 were subclonedinto plasmid pUC19 and analysed by digestion with restrictionendonucleases. Alternatively, the cDNAs were subjected to plasmid rescuefrom lambda Zap using in vivo excision and subsequently analysed bydigestion with restriction endonucleases.

In this way several different clones were identified. Selected antiserafor two of the clones crossreacted with different spots recognised bythe anti-p70 antisera on blots of E. maxima sporozoites separated by 2dPAGE, these clones were then selected for DNA sequence analysis. Thiswas carried out by random subcloning and sequencing using theM13/dideoxynucleotide chain termination method described by Bankier etal. (Techniques in the Life Sciences (Biochemistry) 85: techniques inNucleic Acids Biochemstry 1-34, 1983).

A nucleic acid sequence according to the present invention may beisolated from an Eimeria maxima strain and multiplied by recombinant DNAtechniques including polymerase chain reaction (PCR) technology or maybe chemically synthesized in vitro by techniques known in the art.

A nucleic acid sequence according to the invention can be ligated tovarious replication effecting DNA sequences with which it is notassociated, or linked in nature, resulting in a so-called recombinantvector which can be used for the transformation of a suitable host.Useful recombinant vectors are preferably derived from plasmids,bacteriophages, cosmids or viruses.

Specific vectors or cloning vehicles which can be used to clone nucleicacid sequences according to the invention are known in the art andinclude inter alia plasmid vectors such as pBR322, the various pUC, PGEMand Bluescript plasmids; bacteriophages, e.g. lambdagt-Wes, Charon 28and the M13 derived phages or viral vectors such as SV40, adenovirus orpolyoma virus (see also Rodriguez, R. L. and D. T. Denhardt, ed.,Vectors: A survey of molecular cloning vectors and their uses,Butterworths, 1988; Lenstra, J. A. et al., Arch. Virol., 110, 1-24,1990). The methods to be used for the construction of a recombinantvector according to the invention are known to those of ordinary skillin the art and are inter alia set forth in Maniatis, T. et al.(Molecular Cloning A Laboratory Manual, second edition; Cold SpringHarbor Laboratory, 1989).

For example, the insertion of the nucleic acid sequence according to theinvention into a cloning vector can easily be achieved when both thegenes and the desired cloning vehicle have been cut with the samerestriction enzyme(s) as complementary DNA termini are thereby produced.

Alternatively, it may be necessary to modify the restriction sites thatare produced into blunt ends either by digesting the single-stranded DNAor by filling in the single-stranded termini with an appropriate DNApolymerase. Subsequently, blunt end ligation with an enzyme such as T4DNA ligase may be carried out.

If desired, any restriction site may be produced by ligating linkersonto the DNA termini. Such linkers may comprise specific oligonucleotidesequences that encode restriction site sequences. The restriction enzymecleaved vector and nucleic acid sequence may also be modified byhomopolymeric tailing.

"Transformation", as used herein, refers to the introduction of anheterologous nucleic acid sequence into a host cell, irrespective of themethod used, for example direct uptake or transduction. The heterologousnucleic acid sequence may be maintained through autonomous replicationor, alternatively, may be integrated into the host genome. If desired,the recombinant vectors are provided with appropriate control sequencescompatible with the designated host. These sequences can regulate theexpression of the inserted nucleic acid sequence. In addition tomicroorganisms, cell cultures derived from multicellular organisms mayalso be used as hosts.

The recombinant vectors according to the invention preferably containone or more marker activities that may be used to select for desiredtransformants, such as ampicillin and tetracycline resistance in pBR322,ampicillin resistance and ₋₋ peptide of β-galactosidase in pUC8.

A suitable host cell is a microorganism or cell which can be transformedby a nucleic acid sequence encoding a polypeptide or by a recombinantvector comprising such a nucleic acid sequence, and which can, ifdesired, be used to express said polypeptide encoded by said nucleicacid sequence. The host cell can be of prokaryotic origin, e.g. bacteriasuch as Escherichia coli, Bacillus subtilis and Pseudomonas species; orof eukaryotic origin such as yeasts, e.g. Saccharomyces cerevisiae orhigher eukaryotic cells such as insect, plant or mammalian cells,including HeLa cells and Chinese hamster ovary (CHO) cells. Insect cellsinclude the Sf9 cell line of Spodoptera frugiperda (Luckow et al.,Biotechnology 6, 47-55, 1988). Information with respect to the cloningand expression of the nucleic acid sequence of the present invention ineukaryotic cloning systems can be found in Esser, K. et al. (Plasmids ofEukaryotes, Springer-Verlag, 1986).

In general, prokaryotes are preferred for the construction of therecombinant vectors useful in the present invention. E. coli K12 strainsare particularly useful, especially DH5a or MC1061 strains.

For expression, nucleic acid sequences of the present invention areintroduced into an expression vector, i.e. said sequences are operablylinked to expression control sequences. Such control sequences maycomprise promotors, enhancers, operators, inducers, ribosome bindingsites etc. Therefore, the present invention provides a recombinantvector comprising a nucleic acid sequence encoding an Eimeria proteinidentified above operably linked to expression control sequences, whichis capable of expressing the DNA sequences contained therein in (a)transformed host cell(s).

It should be understood, of course, that the nucleotide sequencesinserted at the selected site of the cloning vector may includenucleotides which are not part of the actual structural gene for thedesired polypeptide, or may include only a fragment of the completestructural gene for the desired protein as long as the transformed hostwill produce a polypeptide having at least one or more immunogenicdeterminants of an Eimeria protein antigen.

When the host cells are bacteria, useful expression control sequenceswhich may be used include the Trp promotor and operator (Goeddel, etal., Nucl. Acids Res., 8, 4057, 1980); the lac promotor and operator(Chang, et al., Nature, 275, 615, 1978); the outer membrane proteinpromotor (Nakamura, K. and Inouge, M., EMBO J., 1, 771-775, 1982); thebacteriophage lambda promotors and operators (Remaut, E. et al., Nucl.Acids Res., 11, 4677-4688, 1983); the ₋₋ amylase (B. subtilis) promotorand operator, termination sequences and other expression enhancement andcontrol sequences compatible with the selected host cell. When the hostcell is yeast, illustrative useful expression control sequences include,e.g., ₋₋ mating factor. For insect cells the polyhedrin or p10 promotersof baculoviruses can be used (Smith, G. E. et al., Mol. Cell. Biol. 3,2156-65, 1983). When the host cell is of mammalian origin illustrativeuseful expression control sequences include the SV-40 promotor (Berman,P. W. et al., Science, 222, 524-527, 1983) or the metallothioneinpromotor (Brinster, R. L., Nature, 296, 39-42, 1982) or a heat shockpromotor (Voellmy et al., Proc. Natl. Acad. Sci. USA, 82, 4949-53,1985). Alternatively, expression control sequences present in Eimeriamay also be applied. For maximizing gene expression, see also Robertsand Lauer (Methods in Enzymology, 68, 473, 1979).

Therefore, the invention also comprises (a) host cell(s) containing anucleic acid sequence or a recombinant nucleic acid molecule or arecombinant vector described above, capable of producing the Eimeriaprotein by expression of the nucleic acid sequence.

Immunization of poultry against Eimeria infection can be achieved byadministering to the birds a protein according to the invention in animmunologically relevant context as a so-called subunit vaccine. Thesubunit vaccine according to the invention may comprise a protein in apure form, optionally in the presence of a pharmaceutically acceptablecarrier. The protein can optionally be covalently bonded to anon-related protein, which can be of advantage in the purification ofthe fusion product. Examples are β-galactosidase, protein A,prochymosine, blood clotting factor Xa, etc.

In some cases the ability to raise protective immunity using theseproteins per se may be low. Small fragments are preferably conjugated tocarrier molecules in order to raise their immunogenicity. Suitablecarriers for this purpose are macromolecules, such as natural polymers(proteins like key hole limpet hemocyanin, albumin, toxins), syntheticpolymers like polyamino acids (polylysine, polyalanine), or micelles ofamphiphilic compounds like saponins. Alternatively these fragments maybe provided as polymers thereof, preferably linear polymers.

If required, the proteins according to the invention which are to beused in a vaccine can be modified in vitro or in vivo, for example byglycosylation, amidation, carboxylation or phosphorylation.

An alternative to subunit vaccines is live vaccines. A nucleic acidsequence according to the invention is introduced by recombinant DNAtechniques into a microorganism (e.g. a bacterium or virus) in such away that the recombinant microorganism is still able to replicate,thereby expressing a polypeptide coded by the inserted nucleic acidsequence and eliciting an immune response in the infected host bird.

A preferred embodiment of the present invention is a recombinant vectorvirus comprising an heterologous nucleic acid sequence described above,capable of expressing the DNA sequence in (a) host cell(s) or host birdinfected with the recombinant vector virus. The term "heterologous"indicates that the nucleic acid sequence according to the invention isnot normally present in nature in the vector virus.

Furthermore, the invention also comprises (a) host cell(s) or cellculture infected with the recombinant vector virus, capable of producingthe Eimeria protein by expression of the nucleic acid sequence.

For example the well known technique of in vivo homologous recombinationcan be used to introduce an heterologous nucleic acid sequence accordingto the invention into the genome of the vector virus.

First, a DNA fragment corresponding with an insertion region of thevector genome, i.e. a region which can be used for the incorporation ofan heterologous sequence without disrupting essential functions of thevector such as those necessary for infection or replication, is insertedinto a cloning vector according to standard recDNA techniques.Insertion-regions have been reported for a large number ofmicroorganisms (e.g. EP 80,806, EP 110,385, EP 83,286, EP 314,569, WO88/02022, WO 88/07088, U.S. Pat. No. 4,769,330 and U.S. Pat. No.4,722,848).

Second, if desired, a deletion can be introduced into the insertionregion present in the recombinant vector molecule obtained from thefirst step. This can be achieved for example by appropriate exonucleaseIII digestion or restriction enzyme treatment of the recombinant vectormolecule from the first step.

Third, the heterologous nucleic acid sequence is inserted into theinsertion-region present in the recombinant vector of the first step orin place of the DNA deleted from said recombinant vector. The insertionregion DNA sequence should be of appropriate length as to allowhomologous recombination with the vector genome to occur. Thereafter,suitable cells can be infected with wild-type vector virus ortransformed with vector genomic DNA in the presence of the recombinantvector containing the insertion flanked by appropriate vector DNAsequences whereby recombination occurs between the corresponding regionsin the recombinant vector and the vector genome. Recombinant vectorprogeny can now be produced in cell culture and can be selected forexample genotypically or phenotypically, e.g. by hybridization,detecting enzyme activity encoded by a gene co-integrated along with theheterologous nucleic acid sequence, or detecting the antigenicheterologous polypeptide expressed by the recombinant vectorimmunologically.

Next, this recombinant microorganisms can be administered to poultry forimmunization whereafter it maintains itself for some time, or evenreplicates in th body of the inoculated animal, expressing in vivo apolypeptide coded for by the inserted nucleic acid sequence according tothe invention resulting in the stimulation of the immune system of theinoculated animal. Suitable vectors for the incorporation of a nucleicacid sequence according to the invention can be derived from virusessuch as pox viruses, e.g. vaccinia virus (EP 110,385, EP 83,286, U.S.Pat. No. 4,769,330 and U.S. Pat. No. 4,722 848) or fowl pox virus (WO88/02022), herpes viruses such as HVT (WO 88/07088) or Marek's Diseasevirus, adeno virus or influenza virus, or bacteria such as E. coli orspecific Salmonella species. With recombinant microorganisms of thistype, the polypeptide synthesized in the host animal can be exposed as asurface antigen. In this context fusion of the polypeptide with OMPproteins, or pilus proteins of for example E. coli or syntheticprovision of signal and anchor sequences which are recognized by theorganism are conceivable. It is also possible that the Eimeriapolypeptide, if desired as part of a larger whole, is released insidethe animal to be immunized. In all of these cases it is also possiblethat one or more immunogenic products will find expression whichgenerate protection against various pathogens and/or against variousantigens of a given pathogen.

A vector vaccine according to the invention can be prepared by culturinga recombinant bacterium or a host cell infected with a recombinantvector comprising a nucleic acid sequence according to the invention,whereafter recombinant bacteria or vector containing cells and/orrecombinant vector viruses grown in the cells can be collected,optionally in a pure form, and formed into a vaccine optionally in alyophilised form.

Host cells transformed with a recombinant vector according to theinvention can also be cultured under conditions which are favourable forthe expression of a polypeptide coded by said nucleic acid sequence.Vaccines may be prepared using samples of the crude culture, host celllysates or host cell extracts, although in another embodiment morepurified polypeptides according to the invention are formed into avaccine, depending on its intended use. In order to purify thepolypeptides produced, host cells transformed with a recombinant vectoraccording to the invention are cultured in an adequate volume and thepolypeptides produced are isolated from such cells, or from the mediumif the protein is excreted. Polypeptides excreted into the medium can beisolated and purified by standard techniques, e.g. salt fractionation,centrifugation, ultrafiltration, chromatography, gel filtration orimmuno affinity chromatography, whereas intra cellular polypeptides canbe isolated by first collecting said cells, disrupting the cells, forexample by sonication or by other mechanically disruptive means such asFrench press, followed by separation of the polypeptides from the otherintracellular components and forming the polypeptides into a vaccine.Cell disruption could also be achieved by chemical (e.g. EDTA ordetergents such as Triton X114) or enzymatic means, such as lysozymedigestion.

Antibodies or antiserum directed against a polypeptide according to theinvention have a potential use in passive immunotherapy, diagnosticimmunoassays and generation of anti-idiotypic antibodies.

The Eimeria proteins as characterized above can be used to produceantibodies, both polyclonal,monospecific and monoclonal. If polyclonalantibodies are desired, techniques for producing and processingpolyclonal sera are known in the art (e.g. Mayer and Walter. eds,Immunochemical Methods in Cell and Molecular Biology, Academic Press,London, 1987). Monospecific antibodies to an immunogen can be affinitypurified from polyspecific antisera by a modification of the method ofHall et al. (Nature, 311, 379-387, 1984). Monospecific antibody, as usedherein, is defined as a single antibody species or multiple antibodyspecies with homogeneous binding characteristics for the relevantantigen. Homogeneous binding, as used herein, refers to the ability ofthe antibody species to bind to a specific antigen or epitope.

Monoclonal antibodies, reactive against the Eimeria proteins accordingto the present invention, can be prepared by immunizing inbred mice bytechniques known in the art (Kohler and Milstein, Nature, 256, 495-497,1975). Hybridoma cells are selected by growth in hypoxanthine, thymidineand aminopterin in an appropriate cell culture medium such as Dulbecco'smodified Eagle's medium. Antibody producing hybridomas are cloned,preferably using the soft agar technique of MacPherson, (Soft AgarTechniques, Tissue Culture Methods and Applications, Kruse and Paterson,eds., Academic Press, 276, 1973). Discrete colonies are transferred intoindividual wells of culture plates for cultivation in an appropriateculture medium. Antibody producing cells are identified by screeningwith the appropriate immunogen. Immunogen positive hybridoma cells aremaintained by techniques known in the art. Specific anti-monoclonalantibodies are produced by cultivating the hybridomas in vitro orpreparing ascites fluid in mice following hybridoma injection byprocedures known in the art.

Anti-idiotypic antibodies are immunoglobulins which carry an "internalimage" of the antigen of the pathogen against which protection isdesired and can be used as an immunogen in a vaccine (Dreesman et al.,J. Infect. Disease, 151, 761, 1985). Techniques for raisinganti-idiotypic antibodies are known in the art (MacNamara et al.,Science, 226, 1325, 1984).

The vaccine according to the invention can be administered in aconventional active immunization scheme: single or repeatedadministration in a manner compatible with the dosage formulation, andin such amount as will be prophylactically effective, i.e. the amount ofimmunizing antigen or recombinant microorganism capable of expressingsaid antigen that will induce immunity in poultry against challenge byvirulent Eimeria parasites. Immunity is defined as the induction of asignificant level of protection in a population of chickens aftervaccination compared to an unvaccinated group.

Next to an increase in protection a vaccine comprising the polypeptideof the invention will also reduce the number of oocysts shedded by theinfected animals. Normally, the shedded oocysts will infect otheranimals in the flock. A decrease in the number of oocysts shedded willthen also give a decrease in the number of animals which is subsequentlyinfected and also a decrease in the number of oocysts shedded will giverise to a lesser infective load.

Furthermore, even without effect on the parasite itself, a vaccine candecrease the incidence of disease. This is especially so when thesymptoms of the disease are caused by products released by the parasite.Vaccines directed against such products alleviate the symptoms withoutattacking the parasite.

For live viral vector vaccines the dose rate per chicken may range from105-108 pfu. A typical subunit vaccine according to the inventioncomprises 1 μg-1 mg of the protein according to the invention. Suchvaccines can be administered intradermally, subcutaneously,intramuscularly, intraperitoneally, intravenously, orally orintranasally.

Additionally the vaccine may also contain an aqueous medium or a watercontaining suspension, often mixed with other constituents in order toincrease the activity and/or the shelf life. These constituents may besalts, pH buffers, stabilizers (such as skimmed milk or caseinhydrolysate), emulsifiers, adjuvants to improve the immune response(e.g. oils, muramyl dipeptide, aluminium hydroxide, saponin, polyanionsand amphipatic substances) and preservatives.

A vaccine comprising the polypeptide of the invention may also compriseother immunogenic proteins of E. maxima or immunogenic proteins of otherEimeria species. Such a combination vaccine will decrease the parasiticload in a flock of poultry and will increase the level of protectionagainst coccidiosis.

It is clear that a vaccine according to the invention may also containimmunogens related to other pathogens of poultry, or may contain nucleicacid sequences encoding these immunogens, like antigens of Marek'sDisease virus (MDV), Newcastle Disease virus (NDV), InfectiousBronchitis virus (IBV), Chicken Anemia Agent (CAA), Reo virus, AvianRetro virus, Fowl Adeno virus, Turkey Rhinotracheitis virus or E. colito produce a multivalent vaccine.

The invention also relates to an "immunochemical reagent", which reagentcomprises a protein according to the invention. The term "immunochemicalreagent" signifies that the protein according to the invention is boundto a suitable support or is provided with a labelling substance.

The supports that may be used are, for example, the inner wall of amicrotest well or a cuvette, a tube or capillary, a membrane, filter,test strip or the surface of a particle such as, for example, a latexparticle, an erythrocyte, a dye sol, a metal sol or metal compound assol particle.

Labelling substances which can be used are, inter alia, a radioactiveisotope, a fluorescent compound, an enzyme, a dye sol, metal sol ormetal compound as sol particle.

A nucleic acid sequence according to the invention can also be used todesign specific probes for hybridization experiments for the detectionof Eimeria related nucleic acids in any kind of tissue.

The present invention also comprises a test kit comprising said nucleicacid sequence useful for the diagnosis of Eimeria infection.

The invention also relates to a test kit to be used in an immunoassay,this test kit containing at least one immunochemical reagent accordingto the invention. The immunochemical reaction which takes place usingthis cest kit is preferably a sandwich reaction, an agglutinationreaction, a competition reaction or an inhibition reaction.

For carrying out a sandwich reaction, the test kit can consist, forexample, of a polypeptide according to the invention bonded to a solidsupport, for example the inner wall of a microtest well, and either alabelled polypeptide according to the invention or a labelledanti-antibody.

The invention is illustrated by the following examples.

EXAMPLE 1 Preparation of antigens of E. maxima sporozoites

1.a.i. Preparation of parasites

Eimeria maxima Houghton strain (E. maxima H) parasites were passagedthrough Light Sussex chickens as described by Long et al. (Folio Vet.Lat., 1976, 6: 201-207). Oocysts were isolated from faeces, sporulatedin 2% potassium dichromate at 29° C. for 72 hours, surface sterilised bywashing in 10% sodium hypochlorite and purified by flotation insaturated sodium chloride. Sporulated oocysts were suspended inphosphate buffered saline (PBS) pH 7.6 and broken by vibration.Sporocysts were suspended in PBS pH 7.6 containing 0.5% w/v porcine bile(Difco) and 0.25% w/v trypsin (Difco 1:250) and incubated at 41° C. for30 minutes. Released sporozoites were washed in PBS pH 8.0, purified oncolumns of DE-52 (Whatman) as described by Schmatz et al. (J.Protozool., 1984, 31: 181-183) and stored as pellets in eppendorf tubesat -70° C.

1.a.ii. Preparation of antigens

Sporozoite pellets (5×107) were solubilised by boiling for 10 minutes in100 ml of sample buffer (50 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 100mM DTT and 10 mg/ml bromophenol blue) then loaded onto a discontinuousSDS-polyacrylamide gel. Gels were electrophoresed and polypeptides weretransferred to nitrocellulose (NC) paper by the method of Towbin andGordon (J. Immunol. Methods, 1984, 72: 313-340). After transfer, the NCpaper was rinsed in PBS pH7.6 containing 0.3% Tween-20 and polypeptideswere visualised by staining with colloidal gold (Aurodye, Cambio,England) according to the manufacturer's instructions.

The NC paper was cut into strips, each of which carried Eimeriapolypeptides of a limited range of molecular mass. Each strip was cutinto small pieces and the pieces transferred to labelled glass vials. Toeach vial, 400 ml of DMSO was added and the mixture left for 60 minutesto ensure solubilisation and sterilisation. NC particles wereprecipitated by the dropwise addition, with vigorous vortexing, of anequal volume of carbonate/bicarbonate buffer (50 mM, pH 9.6). Sampleswere transferred to 1.5 ml microcentrifuge tubes and centrifuged at10,000 g for 5 minutes. NC particles were washed three times in RPMI1640 medium (Gibco Biocult, Paisley, Scotland)), then finally suspendedin 1 ml of this medium, divided into 200 ml aliquots, and stored frozenat -70° C.

EXAMPLE 2 Identification of lymphostimulatory antigens

2.a. Methods

2.a.i. Immunisation of animals

For primary infections, groups of Reaseheath-C chickens (4 weeks old, 10birds per group) were orally dosed with 4000 sporulated oocysts of E.maxima H. For secondary infections, the same birds were orally dosedwith 50,000 sporulated oocysts of E. maxima H. For each experiment anage-matched control group of Reaseheath-C chickens were housedseparately.

2.a.ii. Preparation of peripheral blood lymphocytes

Blood samples (5 ml) were withdrawn from superficial wing veins intoplastic syringes containing heparin (10 units/ml). The blood wastransferred to tubes (Falcon 2027, Becton-Dickinson) and centrifuged at400 rpm for 15 minutes in a Sorvall RC3B centrifuge. The layer of cellsabove the sedimented erythrocytes was carefully removed by pipette intofresh tubes (Falcon 2059, Becton-Dickinson) and centrifuged at 2000 rpmfor 10 minutes. The deposited cells were washed three times in RPMI 1640containing 10% foetal calf serum (FCS, virus and mycoplasma screened,Gibco Biocult), 200 units/ml of penicillin and 200 mg/ml of streptomycin(G. R. Squibb & Sons, Moreton, England) and resuspended in the samemedium at 4×106 cells/ml. 100 ml aliquots of cells (4×105) were pipettedinto round bottomed wells of 96-well plates (Nunc-Gibco, Paisley,Scotland). To each well, 100 ml of a prepared sample was added. Testsamples consisted of prepared NC particle suspensions (see Example1.a.i) diluted in RPMI 1640 medium containing 10% FCS, 200 units/mlpenicillin, 200 mg/ml streptomycin. To prepare dilutions, suspensionswere thawed from -70° C., diluted ten-fold with medium and then atwo-fold dilution series made. Control samples contained NC particlesuspensions devoid of protein diluted identically. A second series ofcontrol samples contained a lysate of whole sporozoites (0.5 mg/ml ofprotein) prepared by freeze-thawing and sonicating sporozoites. Eachsample was prepared in triplicate for each cell preparation withreplicates placed randomly across plates. Plates were incubated for 96hours at 41° C. in 5% CO2, pulsed for the final 16 hours with 1 mCi3H-thymidine at 48 Ci/mMol (Amersham U.K.) then harvested (DynatronMacromash Harvester, Dynatech Laboratories Ltd., Sussex, England) ontoglass microfibre filters (MA781, Dynatron Laboratories Ltd.). Afterdrying for 1 h at 50° C. the discs were placed in scintillation vials,3.5 ml of liquid scintillation cocktail (Scintillator 299Tm Packard,Caversham, U.K.) was added and the radioactive incorporation measured ina scintillation spectrophotometer (Beckman Instruments Inc. LS9000).

2b. Results

Results are expressed as a stimulation index (SI) calculated for eachsample with cells from each bird as follows:

    SI=cpm1/cpm2

where:

cpm1=average counts per minute of triplicate cultures incubated with NCparticles bearing protein.

cpm2=average counts per minute of triplicate cultures incubated with NCparticles devoid of protein.

Solubilised NC strips containing polypeptides with relative molecularmasses of approximately 73.000/71.000/69.000 D (collectively called70.000 D) were found to stimulate the proliferation of lymphocytes frominfected birds (see Table 1.). Lymphocytes from control birds were notstimulated to proliferate. The SIs varied from 4 to 9 and time-coursestudies showed that lymphocytes prepared from birds at 4 days aftersecondary infection proliferated most.

EXAMPLE 3 Raising and screening of antibodies to lymphostimulatoryantigens

3.a. Methods

3.a.i. Immunisation of animals

Pathogen-free rabbits (Harlan-Olac, Bicester, England) were maintainedfree of coccidia. Polypeptides of E. maxima H sporozoite pellets weresolubilised, separated by SDS-polyacrylamide gel electrophoresis andtransferred to NC as described in Example 1. NC strips bearingpolypeptides with molecular masses of 70.000 D were excised, solubilisedin DMSO as described in Example 1, washed in PBS pH 7.0 and finallysuspended in 1 ml of PBS 7.0. Suspensions were injected subcutaneouslyinto 4 sites (0.25 ml per site) and injections were repeated every 2weeks using one NC strip per rabbit each time. Rabbits were bled 2 weeksafter each injection.

3.a.ii. Screening of antisera by one and two dimensional blotting

Polypeptides of E. maxima H sporozoite pellets were solubilised andseparated by SDS-polyacrylamide gel electrophoresis as described inExample 1. Alternatively, sporozoites (7×107) were suspended in 500 mllysis buffer (0.2% Nonidet-P40, 20 mM CHAPS, 9M urea, 0.2% Biolytes 3-10(Biorad), 1 mM DTT), sonicated (three ten-second bursts at 10 microns,MSE soniprep 50) and subjected to three cycles of freeze-thawing.Samples were centrifuged at 12,000 g in a microfuge for 1 minute thenpolypeptides separated by two-dimensional gel electrophoresisessentially as described by O'Farrell (J. Biol. Chem., 1975, 250:4007-4021).

Separated polypeptides were transferred to NC paper as described inExample 1. The NC paper was immersed in TTN buffer (10 mM Tris-HCl pH7.4, 500 mM NaCl, 0.05% Tween-20) containing 3% Bovine serum albumin(BSA) and incubated at room temperature, with gentle rocking, for 2hours. The paper was rinsed in water, cut into strips and each stripincubated for 3 hours in a sample of rabbit serum diluted 1:250 in TTNcontaining 1% BSA. Strips were washed three times in TTN containing 0.5%Tween-20 then incubated for 1 hour in goat anti-rabbit IgG conjugated toalkaline phosphatase (Promega), diluted 1:7500 in TTN containing 1% BSA.Strips were washed a further three times in TTN containing 0.5% Tween-20and once in AP buffer (100 mM Tris pH 9.5, 100 mM NaCl, 10 mM MgCl2).Binding of the phosphatase conjugate was detected by incubating stripsin AP buffer containing 50 mg/ml nitroblue tetrazolium and 50 mg/mlbromochloroindolyl phosphate.

3.b. Results

The specificities of rabbit anti-p70 sera probed onto one-dimensionalWestern blots of polyeptides of E. maxima are shown in FIG. 1.Recognition of spots on two dimensional Western blots is summarised inTable 2.

EXAMPLE 4 Construction of an E. maxima sporozoite cDNA library

4.a. Methods

4.a.i. Isolation of mRNA

E. maxima sporozoites (5×108) were purified as described in Example 1.Total cellular RNA was prepared using an RNA extraction kit (Pharmacia)according to the manufacturer's instructions. Briefly, sporozoites werelysed by incubation in buffered guanidinium thiocyanate, N-laurylsarcosine and EDTA and RNA was separated from other cellular componentsby ultracentrifugation through buffered caesium trifluoroacetate. TheRNA pellet was carefully dissolved in TE buffer (10 mM Tris-HCl pH 7.5,1 mM EDTA) and stored at -70° C. as an ethanol precipitate. MessengerRNA was purified from this total RNA preparation using columns of oligo(dT) cellulose (poly(A) Quik, Stragagene) according to themanufacturer's instructions.

4.a.ii. Synthesis and cloning of cDNA cDNA was synthesised frommessenger RNA using a ZAP-cDNATM synthesis kit (Stratagene) according tothe manufacturer's instructions. The cDNA population ranged in size fromless than 200 bp to around 6 kbp as judged by agarose gelelectrophoresis and autoradiography of a small portion of thesynthesised cDNA. The remaining cDNA was end-repaired using T4 DNApolymerase in the presence of all four dNTPs at 37° C. for 30 minutes.EcoRI adaptors were ligated onto the blunted ends of the cDNA using T4DNA ligase at 8° C. for 24 hours. Digestion with XhoI produced cDNA withXhoI restriction sites at all 3' ends and EcoRI restriction sites at all5' ends. Oligonucleotides (excess adaptors and the restriction enzymedigested primer-template) were removed by centrifuging the samplethrough a 1 ml column of Sephacryl S-400.

100 ng portions of cDNA were ligated to 1 mg Uni-ZAP XR vector(Stratagene, digested with Eco RI and Xho I and dephosphorylated)overnight at 12° C. using T4 DNA ligase. Ligated DNA was packaged intophage heads using Gigapack II Gold packaging extract (Stratagene)according to the manufacturer's protocol. The resulting primarylibraries were plated and amplified on E. coli SURE cells (Stratagene)and the resulting amplified libraries (Emx8 and Emx9) were titred on E.coli XL1-Blue cells (Stratagene) all according to manufacturer'sinstructions. Briefly, for all platings, host cells were grown overnightwith shaking at 30° C. in L Broth supplemented with 0.2% (w/v) maltoseand 10 mM MgSO4. Cells were diluted to OD600=0.5 with 10 mm MgSO4 beforeuse. The number of recombinants in each library was determined byplating phage in the presence of 0.4% (w/v)5-bromo-4-chloro-3-indolyl-β-D-galactoside (Xgal) and 2.5 mMisopropylthio-β-D-galactoside (IPTG) (Northumbria Biologicals Ltd.).

4.b. Results

Emx8 contains 3×108 pfu/ml (65% recombinant) and Emx9 contains 6×108pfu/ml (55% recombinant)

EXAMPLE 5 Identification of cDNA clones coding for E. maxima p70antigens.

5.a. Methods

Immunoscreening of cDNA libraries was done according to standardinstructions supplied by Stratagene. The papers were immersed in rabbitanti-p70 serum diluted 1:100 in TTN containing 1% BSA. All furtherprocedures were identical to those described for the development ofWestern blots in Example 3A. Positive plaques were identified and afterstoring overnight at +4° C. to elute bacteriophage particles, plaqueswere rescreened. Rescreening was continued until all the positivescontained pure populations of antibody reactive plaques.

5.b. Results

Twenty independent plaques (pEm70/1 to pEm70/20) which reacted withrabbit anti-p70 serum were isolated and plaque purified from librariesEmx8 and Emx9.

EXAMPLE 6 Analysis of cDNA clones coding for E. maxima p70 antigens

6.a. Methods

6.a.i. Selection of clone-specific antibodies

0.2 ml aliquots of XL1-Blue cells were infected with 1-3×103 pfu fromplaque purified phage populations. These were plated onto 90 mm dishesand treated according to the procedures described in Example 5.a. up toand including the overnight incubation of NC papers in TTN containing 3%BSA. Papers were then rinsed in TTN, each immersed in 5 ml rabbitanti-p70 serum diluted 1:100 in TTN containing 1% BSA and incubated withgentle rocking at room temperature for 6 hours. Papers were washed threetimes in TTN containing 3% BSA then bound antibody was eluted byimmersing each paper in 5 ml 0.2M glycine pH 2.8 and rocking for 10minutes. The eluant containing clone-specific antibodies was brought toneutral pH by the addition of 0.3M Tris, 10% (w/v) BSA. Clone specificantibodies were used undiluted to probe Western blots of E. maximasporozoite proteins separated by one and two dimensional gelelectrophoresis. All methods for the preparation and immunoscreening ofWestern blots are identical to those described in Example 3.a.ii.

6.a.ii. Analysis of cDNA inserts

DNA was prepared from stocks of recombinant phages using the method ofGrossberger (Nucleic Acids Res., 1987, 15: 6737) and the cDNA insertsreleased by digestion with restriction enzymes Eco RI and Xho I. EachcDNA was ligated to pUC19 (Pharmacia) which had been digested withrestriction enzymes Eco RI and Sal I and dephosphorylated with calfintestinal phosphatase. Ligated DNA was introduced into E. coli strainTG1 by transformation. Plasmids were isolated using the method ofChoudhary (Nucleic Acids Res., 1991, 19: 2792) and analysed by digestionwith restriction endonucleases.

As an alternative to subcloning into plasmid pUC19, cDNA clones wererescued into plasmid pbluescript by in vivo excision from recombinantlambda ZAPII according to instructions supplied by the manufacturer(Stratagene). pBluescript plasmids containing cDNA were isolated byalkaline lysis (Birnboim and Doly, 1979, Nucleic Acids Res., 7: 1513)and cDNAs analysed by digestion with restriction endonucleases.

6.a.iii. DNA sequence determination

pEm70/1 was purified by equilibrium centrifugation in CsCl/ethidiumbromide gradients and the cDNA insert was sequenced by random subcloninginto M13 phage as described by Bankier and Barrell (Techniques in theLife Sciences (Biochemistry) 85: techniques in Nucleic AcidsBiochemistry 1-34, 1983). For other clones, the 5' and 3' ends of thecDNA inserts were sequenced directly from double stranded plasmid DNA.

6.b. Results

6.b.i. Reactivity of clone-specific antibodies

Twenty plaque-purified lambda populations isolated from libraries Emx8and Emx9 (see example 5) were used to produce clone-specific antibodies.Fourteen of these antibodies specifically cross-reacted withpolypeptides of around 70 kDa on one-dimensional blots of E. maximasporozoite proteins. The clone-selected antibodies were used to probetwo dimensional blots of E. maxima sporozoite proteins. Polypeptidespots were identified by arbitrary numbering and the five clone-selectedantibodies reacted with constellations of spots which were sub-sets ofthe spots recognised by intact rabbit anti-p70 serum. This informationis summarised in Table 2.

6.b.ii. Restriction mapping

Analytical restriction enzyme digestions of the fourteen clones thatselected antibodies specific for 70 kDa polypeptides showed there weresix differently sized cDNA inserts. Two of these were called pEm70/1 andpEm70/4. Restriction enzyme maps of the two cDNA inserts are shown inFIG. 2.

6.b.iii. Analysis of DNA sequences

The nucleotide sequence found is of pEm70/1 and its deduced amino acidsequence is shown in SEQ. ID. NO. 1 from nucleic acid 126. The sequencefound in clone pEm70/1 is a 1294 bp cDNA including the 5' EcoRI adaptorand a 3' polyA sequence followed by an XhoI site. The cDNA appears to be"open" from the first triplet of nucleotides to an ochre terminationcodon (TAA) at 1258 bp which precedes the poly(A) sequence. The deducedamino acid sequence encodes a protein of 419 amino acids orapproximately 46 kilodaltons. The cDNA has internal EcoRI sites at 67 bpand 1000 bp and unique ScaI (699 bp), HindIII (736 bp), SspI (995 bp),HincII (1200 bp) and PstI (1251 bp) restriction sites (basepairsmeasured in distance from start of the sequence found in clone pEm70/1,i.e. nucleic acid 126 in SEQ ID NO:1). The analysis of the first 125nucleotides of the sequence shown in SEQ ID NO:1 is discussed in Example9.

The 5' and 3' end sequences of pEm70/4 are shown in SEQ. ID. NOS. 3 and5.

EXAMPLE 7 Expression of the recombinant protein encoded by pEm70/1

7.a. Methods

7.a.i Construction of plasmid pRSETAEm70/1

1 μg of plasmid pEm70/1 was digested with 10 units of BamHI and 10 unitsof XhoI for 2 hours at 37° C. and the cDNA insert ligated intoBamHI-XhoI digested and dephosphorylated plasmid pRSETA (InVitrogen).Ligated DNA was transfected into E. coli strain HMS174, coloniescontaining recombinant plasmid pRSETAEm70/1 were picked and plasmid DNAwas isolated by alkaline lysis (Birnboim and Doly, Nucl. Acid. Res., 7,1513, 1979).

7.a.ii Expression of (His)6-Em70/1 fusion protein from plasmidpRSETAEm70/1.

Bacteria harbouring plasmid were grown overnight in L-broth containing100 μg/ml ampicillin. 10 ml of overnight culture were diluted 1:100 inprewarmed (37° C.) L-broth and grown with aeration for 5 hours at 37° C.Samples were removed for Western blot analysis.

7.a.iii Purification of (His)6-Em70/1 fusion protein by metal-affinitychromatography.

The 5 hour culture of pRSETAEm70/1 was centrifuged and the bacterialpellet resuspended in 5 ml, 6M guanidine-HCl, 20 mM sodium phosphate pH7.8 and sonicated for four bursts of thirty seconds on full power (MSESoniprep). The sonicate was centrifuged at 10,000× g to remove insolublematerial and the supernate mixed with 10 ml immobilised nickel resin("ProBondTM", InVitrogen) which had been equilibrated in 8M urea, 20 mMsodium phosphate pH7.8. The resin and lysate mixture was poured into aglass column and the resin allowed to settle for 30 minutes. A tap atthe bottom of the column was opened and the "flow through" wascollected, re-applied to the resin and collected. The resin was washedwith equilibration buffer until the eluate had an absorbance <0.05 at280 nm (A280). The resin was washed with equilibration buffer at pH6.0to remove non-specifically bound bacterial proteins and the recombinantprotein was finally eluted with equilibration buffer at pH4.0. Therecombinant protein was dialysed for 16 hours at +4° C. against a largevolume of PBS pH7.6. The concentration of protein was estimated using adye binding reagent (Bio-Rad), the yield was approximately 5 mg from 1litre culture.

7.b. Results

Recombinant pEm70/1 was expressed in E. coli as a fusion with sixhistidine residues at its N-terminus. The fusion protein that reactedspecifically with rabbit anti-p70 or with chicken anti-recombinantpEm70/1 sera was a doublet of 46-48 kDa (FIG. 3, track 1). Thecontribution of protein from the expression vector is six histidines andthe enterokinase recognition sequence (asp-asp-asp-lys) which totalsaround 1 kDa. The recombinant protein produced in the 5 hour culture(FIG. 3, track 1) was present in the soluble fraction after sonication(FIG. 3, track 2) and was purified from the nickel affinity column (FIG.3, tracks 4 and 5). FIG. 4 shows the Coomassie stained polyacrylamidegel of the purification of the recombinant protein expressed bypRSETAEm70/1.

EXAMPLE 8 Immunisation of chickens with the recombinant protein(His)6-Em70/1.

8.a. Methods

8.a.i Immunisation of animals.

Thirty six Light Sussex birds were reared under coccidia-free conditionsuntil three weeks of age. Birds were randomly assigned to two groups andwere housed individually in single bird cages. Samples of blood weretaken and eighteen birds were immunised by sub-cutaneous injection (0.1ml) of 25 μg antigen (prepared as described in Example 7) and 100 μgsaponin in PBS. The remaining eighteen birds were immunised with 25 μgbacterial antigen (that did not bind to the nickel resin) and 5 μgsaponin in PBS. Immunisations were repeated twice more at two weeklyintervals and blood samples were taken following each immunisation.

8.a.ii Challenge of animals.

Two weeks after the final immunisation, all birds were given 100sporulated oocysts of E. maxima by oral intubation. The faeces of eachbird were harvested by daily collections onto papered trays and thetotal number of oocysts excreted by each bird from 5 to 10 dayspost-challenge was calculated by counting mixed and diluted samples offaeces in Macmaster counting chambers.

8.b. Results

Table 3 shows the individual oocyst output and group means of the twogroups of birds either immunised with antigen or mock-immunised. Birdswhich received antigen had oocyst outputs lower than the mock-immunisedgroup indicating that the antigen, as described in Example 7, can beused to protect chickens against infections with Eimeria maxima.

EXAMPLE 9 Additional 5' nucleotide sequence of the Em70/1 gene.

9.a. Methods

The 5' RACE (rapid amplification of cDNA ends) technique (Frohman etal., Proc. Natl. Acad. Sci., 85, 8998, 1988; Belyavsky et al. Nucl.Acids Res., 17, 2919, 1988; CLONTECH laboratories) was used to obtainmore of the 5' DNA sequence of the gene encoding the cDNA Em70/1.Messenger RNA was isolated from 200×106 E. maxima sporozoites using aFasttrack kit (InVitrogen). cDNA was synthesized from 2 μg mRNA usingAMV reverse transcriptase according to the manufacturer's instructions(CLONTACH Laboratories Inc) except that 1 μg of random primers was usedto prime first strand cDNA synthesis instead of a gene specific primer.RNA was hydrolysed with NaOH and an anchor primer (SEQ ID NO:6) wasligated to the 5' end of the cDNA using RNA ligase. The ligated cDNA wasdiluted 1:10 with water and then analysed by PCR (polymerase chainreaction) using an anchor specific primer (SEQ ID NO:7) and an antisensegene-specific primer (SEQ ID NO:8). PCR gave a single product ofapproximately 800 bp as judged by agarose gel electrophoresis. Thisfragment was excised from the agarose gel using a GeneClean kit(Stratatech Scientific), digested with 10 units EcoRI and ligated toEcoRI-digested and dephosphorylated M13mp18. After transfection into E.coli strain TG1, recombinant plaques were picked, single stranded DNAwas isolated and the nucleotide sequence of inserts determined using thePRISMTM DyeDeoxyTM Terminator Cycle Sequencing Kit (Applied Biosystems).

9.b. Results

Using the 5' RACE followed by EcoRI sub-cloning, an additional 125nucleotides upstream Em70/1 which linked up with the previously (Example6) determined sequence were obtained.

EXAMPLE 10 Expression of the E. maxima Em70/1 antigen in insect cellsusing baculovirus as vector.

10.a. Construction of pAcEM

The XhoI site of pEm70/1 was converted into BglII by means of asynthetic linker using the methodology as described by Sambrook, J. etal. (Molecular Cloning, A laboratory Manual, Chapter 8). The 1.3 kbinsert of pEm70/1 containing the gene encoding antigen 70/1 wassubsequently isolated as a BamHI-BglII fragment and ligated into theBamHI site of the baculo transfer vector pAcLacZ+MCS obtained from Dr.D. Bishop, Institute of Virology, Oxford, U.K.

Correct integration relative to the polyhedrin promoter was verified byrestriction digests.

From previous sequence analyses it was known that the coding region ofthe insert of pEm70/1 was incomplete at the N-terminus and was lackingan ATG-initiator.

In order to allow expression, a 42 bp synthetic DNA fragment composed ofthe complementary oligos L1 (SEQ ID no. 9) and L2 (SEQ ID no. 10) withBamHI compatible protruding ends, was ligated into the unique BamHI sitethat was restored after insertion of the 1.3 kb fragment from pEm70/1into pAcLacZ+MCS. This resulted in plasmid pAcEM. The correctly inserted42 bp linker in pAcEM provided a reading frame of the first 11 aminoacids of the polyhedrin protein fused to the E. maxima gene encoding amajor part of antigen 70/1. The final structure downstream of thepolyhedrin promoter in plasmid pAcEM was verified by nucleotidesequencing.

10.b. Transfection of insect cells and isolation of baculovirusrecombinants.

pAcEM DNA was transfected into Sf9 cells using standard techniques (D.O'Reilly, K. Miller, and V. Luckow: Baculovirus expression vectors, alaboratory manual, Oxford University press, 1994), using Lipofectin(Gibco/BRL) and wildtype baculovirus DNA. Recombinant plaques wereidentified on tissue-culture dishes with X-gal (Gibco/BRL) in theagar-overlay. Blue plaques were picked and replated three times, till nowildtype virus was detectable. The viral plaque was amplified in T75 andT175 flasks and finally in a 100 ml spinnerbottle with SF9 cells. Theviral titre was determined by way of immunofluorescence: microtitreplates with Sf9 cells were infected with a dilution of the virus. After6 days of incubation the plates were fixed with alcohol, and stainedwith a 1:500 dilution of a chicken polyclonal antibody raised againstthe Em70/1 sporozoite protein. Next incubation was with 1:600Goat-anti-chicken IgG (KPL)-conjugated to FITC. The titre of the stockof vAcEM virus was 7.2 Log10 TCID50/ml.

10.c. Production of E. maxima 70/1 protein in insect cells.

Four T175 flasks each containing 3×107 Sf9 cells in exponential growthwere infected with the vAcEM virus stock at an moi of 10 TCID50/cell.After three days the infected cells were harvested on ice. The cellswere resuspended at 1×107/ml in PBS and sonicated. The sonicates wereinactivated with 0.075% formalin, by incubating at 4° C. for 1 week.Formaldehyde was neutralized with an equimolar amount of Na2S205.

10.d. Analysis of baculo-expressed product using SDS-PAGE and WesternBlotting.

Samples of resuspended cells after sonication were mixed 2:3 withreduced (R) or non-reduced (NR) sample buffer (consisting of 188 mMTris-HCl pH6.8, 6% SDS±0.2M β-mercapto-ethanol), boiled for 10 min. at95° C., centrifuged for 10 min at 13000 rpm and loaded onto SDS-gels.Uninfected cells and SHAM-recombinant infected cells without insert wereincluded as controls.

Samples were analysed on 12% PAA gel (1.5 mm thick). In each lane 80 μlsample was loaded. Part of the gel was stained with CBB (CoommassieBrilliant Blue R250), the rest was electro-blotted onto nitrocellulose.

Blot was incubated with chickenserum raised against E. max 70/1sporozoite protein diluted 1:500 (or negative chicken serum as control).Both blots were developed with alkaline phosphatase-labelled goatanti-chicken IgG and the NBT/BCIP substrate according to standardprotocols.

Only the Em70/1 containing virus expressed a band recognised by thechicken serum. The approximate Mr of the expressed product was 45,000,with a break-down product visible at Mr 33,000. No difference wasdetectable in expression and recognition after running the gel underreducing or non-reducing conditions.

EXAMPLE 11 Immunisation of chickens with Salmonella gallinarum-Em70/1

11.a. Methods

2 μg of purified plasmid pEm70/1 was cloned into the MCS (multiplecloning site) of pMLB1113(His)6₋₋ 3 vector (see FIG. 5.) after digestionwith EcoRI (20 units). The ligated DNA was used to transform E. coliTG1. Recombinant colonies were selected on L-agar plates containing 100μg ampicillin and plasmids prepared by alkaline lysis (Birnboim & Doly).Plasmids were analysed by restriction enzyme digestion and agarose gelelectrophoresis and a single recombinant plasmid pMLB(His)6₋₋ 3-Em70/1,containing the 932 bp EcoRI fragment in the right orientation wasselected.

Salmonella gallinarum 9R strain (H. Williams Smith, J. Hygiene, 54, 419,1956) was transformed with plasmid pMLB1113(His)6₋₋ 3-Em70/1 using thehigh effiency protocol of Hanahan (J. Mol. Biol. 166, 557, 1983). Oneplaque purified recombinant colony was cultured, with aeration, in 50 mlL-broth+50 μg ampicillin at 37° C. and grown to a density of 108bacteria/ml.

The bacteria were concentrated and washed by centrifugation for 10 minat 4000 g at 4° C. and diluted with L-broth to the immunisingconcentration being 109/ml.

Twelve 3 wk old White-Leghorn chickens were injected with 108 Salmonella9R-Em70/1 in 0.1 ml L-broth given intramuscularly. This vaccination wasrepeated twice with two week intervals. Salmonella 9R parent strain wasprocessed similarly and given in the same dose to control chickens. Allchickens were challenged at two weeks after the final vaccination.Challenge and assessment of protection was performed as described inexample 8.a.ii.

11.b. Results

The reduction in mean oocyst output of both groups is given in Table 4.

Birds which received the Salmonella vector vaccine had on average loweroocyst outputs compared to their controls. This indicates that thisvector vaccine could be used to reduce the parasite burden of vaccinatedanimals and thereby the pathogenic effects associated with theinfection.

                  TABLE 1                                                         ______________________________________                                        Stimulation Indices of Immune and Control birds                               exposed to nitrocellulose-bourne antigents of E. maxima                       sporozoites. Each S.I. is the mean of ten individual                          birds.                                                                        Strip                        Experiment                                                                            Experiment                               number Experiment 1                                                                             Experiment 2                                                                             1       2                                        (Mr)   Immune birds                                                                             Immune birds                                                                             Control birds                                                                         Control birds                            ______________________________________                                        6 (73k)                                                                              3.12       2.60       1.11    1.04                                     7 (71k)                                                                              2.60       1.97       1.39    1.16                                     8 (69k)                                                                              5.57       4.76       0.99    1.64                                     ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        2-D gel analysis of E. maxima H sporozoites -                                 indentification of spots which are lymphostimulatory,                         recognised by rabbit anti-70kDa serum or recognized by                        clone-specific antibodies.                                                                          Clone 12                                                Spot no.                                                                              anti-70 kDa sera                                                                            pEM70/1  lympho-stimulatory                             ______________________________________                                         5      +                                                                      6      +             +                                                        7      +                                                                     10      +                                                                     13      +                      +                                              22      +             +                                                       23      +             +        +                                              41      +             +                                                       42      +             +                                                       43      +             +                                                       48      +             +        +                                              52      +                                                                     54      +             +                                                       56      +                                                                     ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        In vivo protection against challange infection                                with E. maxima: Oocyst outputs of birds immunised with                        fusion protein pRSETAEm70/1 (group 1) compared to                             mock-immunised birds (group 2).                                                                 Oocyst output                                               Group    Bird No. (×10.sup.6)                                                                           mean SD                                       ______________________________________                                        1         1       37.9          33.4 14.8                                               3       50.5                                                                  4       30.0                                                                  5       12.4                                                                  6       44.2                                                                  7       48.5                                                                  8       28.1                                                                  9       37.9                                                                 10       46.7                                                                 11       42.9                                                                 12       27.2                                                                 13       36.8                                                                 14       22.6                                                                 15       61.3                                                                 16       12.0                                                                 17       14.0                                                                 18       15.6                                                        2        19       33.3          40.4 12.7                                              20       36.8                                                                 21       18.8                                                                 22       36.5                                                                 23       48.2                                                                 24       30.3                                                                 25       48.6                                                                 26       29.4                                                                 27       32.5                                                                 28       43.5                                                                 29       48.0                                                                 30       41.0                                                                 31       20.4                                                                 32       41.6                                                                 33       39.0                                                                 34       49.9                                                                 35       61.0                                                                 36       68.9                                                        ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Mean E. maxima oocyst output of chickens                                      vaccinated with S. gallinarum-Em70/1                                          Treatment       Oocyst output  ×10-6!                                   ______________________________________                                        S. gallinarum-Em70-1                                                                          34.0 ± 9                                                   S. gallinarum 9R control                                                                      41.3 ± 13                                                  ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1400 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: C-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Eimeria maxima                                                  (B) STRAIN: Houghton                                                          (D) DEVELOPMENTAL STAGE: sporozoite                                           (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: sporozoite cDNA cloned in Lambda ZAPII                           (B) CLONE: Em70-1                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1368                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAATTCGTGGAAGTTTTGGGTGAGGTCATCTTATGCAAGGACAAGATA48                            GluPheValGluValLeuGlyGluValIleLeuCysLysAspLysIle                              151015                                                                        ACAGCACAGGAATATGCAGTAAAAGTAATATCTAAACGTCAAGTAAAA96                            ThrAlaGlnGluTyrAlaValLysValIleSerLysArgGlnValLys                              202530                                                                        CAGAAGACAGATAAAGAATTATTATTAAAAGAAGTTGAATTATTAAAG144                           GlnLysThrAspLysGluLeuLeuLeuLysGluValGluLeuLeuLys                              354045                                                                        AAATTAGATCATCCTAATATCATGAAATTATATGAATTCTTTGAGGAT192                           LysLeuAspHisProAsnIleMetLysLeuTyrGluPhePheGluAsp                              505560                                                                        AAAGGATACTTTTATCTTGTTACAGAAGTATATACAGGAGGAGAATTA240                           LysGlyTyrPheTyrLeuValThrGluValTyrThrGlyGlyGluLeu                              65707580                                                                      TTTGATGAAATTATTAATCGAAAAAGATTCAGCGAGGCGGATGCAGCT288                           PheAspGluIleIleAsnArgLysArgPheSerGluAlaAspAlaAla                              859095                                                                        CGTATAGTACGTCAGGTTCTATCGGGTATAAATTATATGCATCGTAAT336                           ArgIleValArgGlnValLeuSerGlyIleAsnTyrMetHisArgAsn                              100105110                                                                     AAAATAGTTCATAGAGATTTAAAGCCAGAGAATTTATTATTAGAGAAT384                           LysIleValHisArgAspLeuLysProGluAsnLeuLeuLeuGluAsn                              115120125                                                                     AAAAAAAAAGATGCAAATATACGAATTATTGATTTTGGGTTATCTACA432                           LysLysLysAspAlaAsnIleArgIleIleAspPheGlyLeuSerThr                              130135140                                                                     CATTTTGAGCCCCAAAAAAAAATGAAGGATAAAATCGGGACCGCGTAC480                           HisPheGluProGlnLysLysMetLysAspLysIleGlyThrAlaTyr                              145150155160                                                                  TACATTGCCCCTGAGGTGCTGCACGGAACATACGATGAGAAATGCGAC528                           TyrIleAlaProGluValLeuHisGlyThrTyrAspGluLysCysAsp                              165170175                                                                     GTCTGGTCTACGGGTGTTATCCTCTATATCCTTCTCTCTGGTTGTCCT576                           ValTrpSerThrGlyValIleLeuTyrIleLeuLeuSerGlyCysPro                              180185190                                                                     CCATTTAACGGAGCAAATGAATTTGAAATTCTAAAGAAAGTCGAGAAA624                           ProPheAsnGlyAlaAsnGluPheGluIleLeuLysLysValGluLys                              195200205                                                                     GGAAAATTCACCTTCGATTTACCACAGTGGCGTAAGGTTAGCGAGCCA672                           GlyLysPheThrPheAspLeuProGlnTrpArgLysValSerGluPro                              210215220                                                                     GCAAAAGATTTAATTAGGAAGATGTTAGCATATGTACCCTCAATGCGT720                           AlaLysAspLeuIleArgLysMetLeuAlaTyrValProSerMetArg                              225230235240                                                                  ATATCAGCAAAAGATGCATTAGATCATCCATGGATAAAAAGTACAGAT768                           IleSerAlaLysAspAlaLeuAspHisProTrpIleLysSerThrAsp                              245250255                                                                     GTTACTGCTAAGGATAGTATTAATCTTCCTTCTCTTGAGAGTACTATA816                           ValThrAlaLysAspSerIleAsnLeuProSerLeuGluSerThrIle                              260265270                                                                     CTTAATATCAGGCAGTTCCAGGGTACACAGAAGCTTGCTGCTGCTGCT864                           LeuAsnIleArgGlnPheGlnGlyThrGlnLysLeuAlaAlaAlaAla                              275280285                                                                     CTGCTGTACATGGGGAGTAAATTAACAACAAATGAGGAGACAGACGAA912                           LeuLeuTyrMetGlySerLysLeuThrThrAsnGluGluThrAspGlu                              290295300                                                                     TTGAATAAAATCTTCCAGAAGATGGATAAGAACGGAGACGGACAACTC960                           LeuAsnLysIlePheGlnLysMetAspLysAsnGlyAspGlyGlnLeu                              305310315320                                                                  GATAAACAAGAATTAATGGAGGGTTATGTTGAATTAATGAAGCTAAAA1008                          AspLysGlnGluLeuMetGluGlyTyrValGluLeuMetLysLeuLys                              325330335                                                                     GGAGAAGATGTTTCTGTATTAGACAAGAGTGCAATTGAGACAGAAGTT1056                          GlyGluAspValSerValLeuAspLysSerAlaIleGluThrGluVal                              340345350                                                                     GAACAAGTTCTTGAGGCTGTAGACTTCGATAAGAATGGATTTATTGAA1104                          GluGlnValLeuGluAlaValAspPheAspLysAsnGlyPheIleGlu                              355360365                                                                     TATTCAGAATTCGTGACGGTGGCAATGGATAGAAGAACTCTGTTATCA1152                          TyrSerGluPheValThrValAlaMetAspArgArgThrLeuLeuSer                              370375380                                                                     AGACAAAGACTTGAAAGAGCATTCGAGATGTTCGACTCGGATGGATCA1200                          ArgGlnArgLeuGluArgAlaPheGluMetPheAspSerAspGlySer                              385390395400                                                                  GGAAAAATCTCCTCCTCTGAATTAGCTACTATATTTGGTGTAAGCGAG1248                          GlyLysIleSerSerSerGluLeuAlaThrIlePheGlyValSerGlu                              405410415                                                                     TTAGACTCGGAGGCATGGCGTCGCGTATTAGCAGAAGTTGATCGAAAT1296                          LeuAspSerGluAlaTrpArgArgValLeuAlaGluValAspArgAsn                              420425430                                                                     AATGACGGAGAAGTTGACTTTGAGGAATTTCAGCAAATGCTTCTTAAA1344                          AsnAspGlyGluValAspPheGluGluPheGlnGlnMetLeuLeuLys                              435440445                                                                     TTATGTGGTAATACTGCAGCAGAATAAATAAATAAATAAAAAAAAAAAAAAAAA1398                    LeuCysGlyAsnThrAlaAlaGlu                                                      450455                                                                        AA1400                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 456 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GluPheValGluValLeuGlyGluValIleLeuCysLysAspLysIle                              151015                                                                        ThrAlaGlnGluTyrAlaValLysValIleSerLysArgGlnValLys                              202530                                                                        GlnLysThrAspLysGluLeuLeuLeuLysGluValGluLeuLeuLys                              354045                                                                        LysLeuAspHisProAsnIleMetLysLeuTyrGluPhePheGluAsp                              505560                                                                        LysGlyTyrPheTyrLeuValThrGluValTyrThrGlyGlyGluLeu                              65707580                                                                      PheAspGluIleIleAsnArgLysArgPheSerGluAlaAspAlaAla                              859095                                                                        ArgIleValArgGlnValLeuSerGlyIleAsnTyrMetHisArgAsn                              100105110                                                                     LysIleValHisArgAspLeuLysProGluAsnLeuLeuLeuGluAsn                              115120125                                                                     LysLysLysAspAlaAsnIleArgIleIleAspPheGlyLeuSerThr                              130135140                                                                     HisPheGluProGlnLysLysMetLysAspLysIleGlyThrAlaTyr                              145150155160                                                                  TyrIleAlaProGluValLeuHisGlyThrTyrAspGluLysCysAsp                              165170175                                                                     ValTrpSerThrGlyValIleLeuTyrIleLeuLeuSerGlyCysPro                              180185190                                                                     ProPheAsnGlyAlaAsnGluPheGluIleLeuLysLysValGluLys                              195200205                                                                     GlyLysPheThrPheAspLeuProGlnTrpArgLysValSerGluPro                              210215220                                                                     AlaLysAspLeuIleArgLysMetLeuAlaTyrValProSerMetArg                              225230235240                                                                  IleSerAlaLysAspAlaLeuAspHisProTrpIleLysSerThrAsp                              245250255                                                                     ValThrAlaLysAspSerIleAsnLeuProSerLeuGluSerThrIle                              260265270                                                                     LeuAsnIleArgGlnPheGlnGlyThrGlnLysLeuAlaAlaAlaAla                              275280285                                                                     LeuLeuTyrMetGlySerLysLeuThrThrAsnGluGluThrAspGlu                              290295300                                                                     LeuAsnLysIlePheGlnLysMetAspLysAsnGlyAspGlyGlnLeu                              305310315320                                                                  AspLysGlnGluLeuMetGluGlyTyrValGluLeuMetLysLeuLys                              325330335                                                                     GlyGluAspValSerValLeuAspLysSerAlaIleGluThrGluVal                              340345350                                                                     GluGlnValLeuGluAlaValAspPheAspLysAsnGlyPheIleGlu                              355360365                                                                     TyrSerGluPheValThrValAlaMetAspArgArgThrLeuLeuSer                              370375380                                                                     ArgGlnArgLeuGluArgAlaPheGluMetPheAspSerAspGlySer                              385390395400                                                                  GlyLysIleSerSerSerGluLeuAlaThrIlePheGlyValSerGlu                              405410415                                                                     LeuAspSerGluAlaTrpArgArgValLeuAlaGluValAspArgAsn                              420425430                                                                     AsnAspGlyGluValAspPheGluGluPheGlnGlnMetLeuLeuLys                              435440445                                                                     LeuCysGlyAsnThrAlaAlaGlu                                                      450455                                                                        (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 242 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (v) FRAGMENT TYPE: C-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Eimeria maxima                                                  (B) STRAIN: Houghton                                                          (D) DEVELOPMENTAL STAGE: Sporozoite                                           (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: sporozoite cDNA cloned in Lambda ZAPII                           (B) CLONE: Em70-4, 5'end of clone                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..242                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      GTGCTATTGCAGGTCTTAATGTTATTCGTATTATTAATGAACCTACT47                             AlaIleAlaGlyLeuAsnValIleArgIleIleAsnGluProThr                                 151015                                                                        GCTGCTGCTATTGCTTACGGTCTTGATAAAAAAGACGAAAAGACTATC95                            AlaAlaAlaIleAlaTyrGlyLeuAspLysLysAspGluLysThrIle                              202530                                                                        CTTGTCTACGATCTTGGTGGTGGTACCTTTGATGTATCCGTCCTTGTT143                           LeuValTyrAspLeuGlyGlyGlyThrPheAspValSerValLeuVal                              354045                                                                        ATTGACAACGGTGTATTCGAAGTCCATGCAACTTCAGGTGATACACAT191                           IleAspAsnGlyValPheGluValHisAlaThrSerGlyAspThrHis                              505560                                                                        CTAGGAGGAGAAGATTTCGATCAGAGAGTAATGGATCACTTCCTGAAG239                           LeuGlyGlyGluAspPheAspGlnArgValMetAspHisPheLeuLys                              657075                                                                        ATT242                                                                        Ile                                                                           80                                                                            (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      AlaIleAlaGlyLeuAsnValIleArgIleIleAsnGluProThrAla                              151015                                                                        AlaAlaIleAlaTyrGlyLeuAspLysLysAspGluLysThrIleLeu                              202530                                                                        ValTyrAspLeuGlyGlyGlyThrPheAspValSerValLeuValIle                              354045                                                                        AspAsnGlyValPheGluValHisAlaThrSerGlyAspThrHisLeu                              505560                                                                        GlyGlyGluAspPheAspGlnArgValMetAspHisPheLeuLysIle                              65707580                                                                      (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 250 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Eimeria maxima                                                  (B) STRAIN: Houghton                                                          (D) DEVELOPMENTAL STAGE: Sporozoite                                           (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: sporozoite cDNA cloned in Lambda ZAPII                           (B) CLONE: Em70-4, 3'end of clone                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      TTTTTTTTTTTTTTTTTTCTATCCTCATCTGCAAACTTTTCTGCTTCTTGTATCTACCTT60                ACTATCTCATCAGGAGTAAGTCTTCCTTATCATTAGTTATTGTTATTTTCTCGCTCTTTC120               CTGTTCCTTTATCTACAGCACTTACATTCAATATACCGTTCCTGTCTACATCAAATGTTA180               CATCTATCTGTGGTACACCACGAGGTGCAGGAGGTATTCCTGTTAATTCAAACTTTCCTA240               ATAAATGGTT250                                                                 (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CACGAATTCACTATCGATTCTGGAACCTTCAGAGG35                                         (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CTGGTTCGGCCCACCTCTGAAGGTTCCAGAATCGATAG38                                      (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TATACCCGATAGAACCTGACG21                                                       (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GATCTAAATATGCCGGATTATTCATACCGTCCCACCATCGGTACCG46                              (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GATCCGGTACCGATGGTGGGACGGTATGAATAATCCGGCATATTTA46                              __________________________________________________________________________

We claim:
 1. A nucleic acid molecule that codes for a protein that has asequence selected from the croup consisting of SEQ ID NO:2, SEQ ID NO:4and portions of SEQ ID NO:2 and SEQ ID NO:4 that comprise an epitopethat will bind to antibodies in antiserum raised against either of thepeptides of SEQ ID NO:2 and SEQ ID NO:4.
 2. A nucleic acid moleculeaccording to claim 1 wherein the nucleic acid molecule contains at leastpart of the DNA sequence shown in SEQ ID No.
 3. 3. A recombinant nucleicacid molecule comprising a nucleic acid molecule according to claim 1operatively linked to expression control sequences enabling expressionof said nucleic acid molecule.
 4. A recombinant vector comprising anucleic acid molecule according to claim
 1. 5. A recombinant vectoraccording to claim 4 wherein the nucleic acid molecule is operativelylinked to expression control sequences.
 6. A host cell or organismtransformed with a recombinant vector molecule according to claim
 4. 7.A process for expression of an Eimeria protein, comprising culturing ahost cell according to claim
 6. 8. A host cell or organism transformedwith a nucleic acid molecule according to claim
 1. 9. A process forexpressing an Eimeria protein comprising culturing a host cell accordingto claim
 8. 10. A nucleic acid molecule according to claim 1 wherein thenucleic acid molecule contains the DNA sequence shown in SEQ ID No. 1 ora portion thereof that codes for an epitope that will bind to antibodiesof antisera raised against the protein that is coded for by SEQ ID NO:1.11. A recombinant nucleic acid molecule comprising a nucleic acidmolecule according to claim 10 operatively linked to expression controlsequences enabling expression of said nucleic acid molecule.
 12. Arecombinant vector comprising a nucleic acid molecule according to claim10.
 13. A recombinant vector according to claim 12, wherein the nucleicacid molecule is operatively linked to expression control sequences. 14.A host cell or organism transformed with a recombinant vector accordingto claim
 12. 15. A process for expression of an Eimeria proteincomprising culturing a host cell according to claim
 14. 16. A host cellor organism transformed with a nucleic acid molecule according to claim10.
 17. A process for expressing an Eimeria protein comprising culturinga host cell according to claim 16.